Cancer metastasis involves a series of mechanical events at the single-cell level. In order to invade to distal sites, aggressive cells must be able to squeeze across small spaces in the extracellular matrix (ECM) of the tumor stroma and endothelial barrier and circulate and traffic through microvessels smaller than the size of the cell (A. F. Chambers et al., Nature Reviews Cancer, 2002, 2, 563-572; P. Friedl et al., Nature Reviews Cancer, 2003, 3, 363-374; P. Friedl et al., Current Opinion in Cell Biology, 2011, 23, 55-64). Under such confined microenvironments, these cells must acquire deformed morphologies. There have been many studies on cell deformability, with techniques ranging from more conventional atomic force microscopy (AFM) (M. J. Rosenbluth et al., Biophysical Journal, 2006, 90, 2994-3003; M. P. Stewart et al., Nat. Protocols, 2012, 7, 143-154) and micropipette aspiration (MPA) (R. M. Hochmuth, Journal of Biomechanics, 2000, 33, 15-22) to more recent microfluidic systems with active (optical forces, hydrodynamic inertial focusing) (J. Guck et al., Biophysical Journal, 2005, 88, 3689-3698; I. Sraj et al., Journal of Biomedical Optics, 2010, 15, 047010-047010; D. R. Gossett et al., Proc. Natl. Acad. Sci. USA, 2012, 109, 7630-7635) and passive (microconstrictions) (S. Gabriele et al., Biophysical Journal, 2009, 96, 4308-4318; A. Adamo et al., Analytical Chemistry, 2012, 84, 6438-6443; W. Zhang et al., Proceedings of the National Academy of Sciences, 2012, 109, 18707-18712) deformation actuators. In particular, we are interested in deformations in the most extreme form observed in physiological systems—deformations at the subnucleus scale. This is important because such deformations are often observed in cell invasion through the ECM and in microcirculation (P. Friedl et al., Current Opinion in Cell Biology, 2011, 23, 55-64; K. Yamauchi et al., Cancer Research, 2005, 65, 4246-4252; P. Friedl et al., Nat Rev Mol Cell Biol, 2012, 13, 743-747; A. Pathak et al., Integrative Biology, 2011, 3, 267-278). These events in the metastatic process suggest that cell deformability is an important property in the context of cancer.
Recent work using microfluidic techniques has shown that deformability may be correlated with disease states in cells, metastatic potential, and stem cell differentiation (J. Guck et al., Biophysical Journal, 2005, 88, 3689-3698; D. R. Gossett et al., Proc. Natl. Acad. Sci. USA, 2012, 109, 7630-7635; W. Zhang et al., Proceedings of the National Academy of Sciences, 2012, 109, 18707-18712). Deformability in these cases is often measured by the aspect ratio of a cell under a fixed stress, such that more deformable cells exhibit a higher aspect ratio. Another common metric is the amount of time it takes a cell to flow through a microconstriction under pressure. These assays are typically high throughput and automated (have minimal manual operations) during measurements, which offer appeal towards clinical applications.
A key disadvantage of these high throughput microfluidic assays is that the information content is typically simplistic and does not fully appreciate the complexity of a biological phenomenon. In particular, the mechanical properties of cells are intrinsically complex in nature and heterogeneous. Not only does heterogeneity exist between different components of the cell, such as the cytoplasm, cytoskeleton, and nucleus, but heterogeneity exists even within the cytoskeletal and nucleoskeletal networks. As a result, a simple one-shot measurement of each cell (i.e. aspect ratio under asymmetric stress or average transit time across a barrier), while offering an appealing and simple assay, is a reductionist characterization of biological cells. Fundamental properties, such as creep strain dynamics, that are pertinent to the deformability of viscoelastic materials are difficult to measure with such techniques. As such, conventional, high resolution and more comprehensive measurements from traditional techniques such as AFM and MPA offer more detailed information about the state and fundamental properties of individual cells.
Micropipette aspiration and atomic force microscopy have been used to elucidate more complex phenomena associated with the mechanical properties of cells and nuclei. For instance, micropipette studies were able to produce high resolution data that revealed and enabled the development of mathematical models of the viscoelasticity of different cell types, which as an example characterized the distinction between solid like cells (endothelial cells) and liquid like cells (neutrophils) (R. M. Hochmuth, Journal of Biomechanics, 2000, 33, 15-22). Additionally, MPA of isolated cell nuclei identified the contributions of different subnucleus structures on force bearing properties under different conditions (swollen and unswollen nuclei) and further revealed that the creep compliance of the nucleus follows a power-law temporal dependence over time scales from 0.1 to 1000 seconds (K. N. Dahl et al., Biophysical Journal, 2005, 89, 2855-2864). AFM studies have also been critical in revealing local cell stiffness as well as cell forces and stress under compression and extension (M. P. Stewart et al., Nat. Protocols, 2012, 7, 143-154; D. A. Fletcher et al., Nature, 2010, 463, 485-492).
In these existing methods, there is a tradeoff between 1) experimental simplicity and automation and 2) the complexity of the measurable properties. More complex material properties such as cell strain dynamics during deformation and relaxation require more complicated procedures that are practicable typically only in labor intensive and bulky apparatuses (MPA and AFM) (M. J. Rosenbluth et al., Biophysical Journal, 2006, 90, 2994-3003; M. P. Stewart et al., Nat. Protocols, 2012, 7, 143-154; R. M. Hochmuth, Journal of Biomechanics, 2000, 33, 15-22), while more automated systems such as microfluidic constriction assays, optical stretchers, and inertial focusing methods produce static and reductionist measurements and are currently limited to simple experimental procedures (J. Guck et al., Biophysical Journal, 2005, 88, 3689-3698; I. Sraj et al., Journal of Biomedical Optics, 2010, 15, 047010-047010; D. R. Gossett et al., Proc. Natl. Acad. Sci. USA, 2012, 109, 7630-7635; S. Gabriele et al., Biophysical Journal, 2009, 96, 4308-4318; A. Adamo et al., Analytical Chemistry, 2012, 84, 6438-6443). The incorporation of more functionality in microfluidic assays often requires more manual labor or additional components such as robotic actuators for image-assisted flow modulation, thus reducing their automation or adding to their already bulky systems that require external pressure pumps and optical components (e.g. high power lasers). These tradeoffs limit the adoptability of the mentioned techniques and thus the practicability of the field of cell biomechanics to select experts in select settings. Mechanical properties such as cell deformability and viscoelasticity, however, are critical and complementary to many areas in cell biology, with implications in cancer metastasis, immune cell responses, tissue homeostasis, blood diseases, and stem cell differentiation (D. A. Fletcher et al., Nature, 2010, 463, 485-492; D. Discher et al., Annals of biomedical engineering, 2009, 37, 847-859; F. Lautenschläger et al., Proceedings of the National Academy of Sciences, 2009, 106, 15696-15701; S. Kumar et al., Cancer and Metastasis Reviews, 2009, 28, 113-127; M. J. Paszek et al., Cancer Cell, 2005, 8, 241-254; Y. Park et al., Proceedings of the National Academy of Sciences, 2010, 107, 6731-6736; D. A. Fedosov et al., Proceedings of the National Academy of Sciences, 2011, 108, 35-39; W. H. Grover et al., Proceedings of the National Academy of Sciences, 2011, 108, 10992-10996; J. P. Shelby et al., Proceedings of the National Academy of Sciences, 2003, 100, 14618-14622). Therefore there is a need for multifunctional, procedurally adept, and automated systems that require minimal labor and components in order to promote accessibility and technology adoption.
The present invention is directed to overcoming these and other deficiencies in the art.